The present invention relates to inhibitors of the oncogenic protein kinases, including ALK, RET and Bcr-Abl. The formula of the inhibitors is disclosed below (Formula I). Such inhibitors can be used for the treatment of hyper-proliferative diseases such as cancer, in particular for the treatment of ALK fusion protein positive cancers, such as anaplastic large cell lymphoma (ALCL), diffuse large B cell lymphoma (DLBCL), inflammatory myofibroblastic tumours (IMT) and non-small cell lung cancer (NSCLC), as well as T315I Bcr-Abl positive cancers such as Chronic Myeloid Leukemia (CML) and Ph+ Acute lymphoblastic leukemia (ALL), and thyroid cancer linked to RET, such as papillary thyroid carcinoma (PTC) and multiple endocrine neoplasia type 2 (MEN2).
Cancer results from the subversion of processes that control the normal growth, location and mortality of cells. This loss of normal control mechanisms arises from the acquisition of mutations that lead to the oncogenic activation of proteins that are involved in the normal regulation of such processes.
Protein kinases are enzymes that catalyse the transfer of phosphate from adenosine-5′-triphosphate (ATP) to specific amino acid residues in many proteins. Generally, the phosphorylation of a protein changes its functionality, from inactive to active in some cases, and from active to inactive in others. Protein kinases are thus involved in the regulation of many aspects of cell function, as most of the signal transduction pathways controlling cell growth, survival, differentiation and motility are mediated by phosphorylation. Abnormal activity of protein kinases has been implicated in many cancers as well as in other diseases. The human genome encodes at least 518 kinases, of which approximately 90 specifically phosphorylate the phenolic hydroxyl of tyrosine residues. Tyrosine kinases are particularly involved in cell proliferation and survival processes, and their aberrant activation most often leads to oncogenic transformation.
For example, structural alterations in ALK produced by the chromosomal rearrangement t(2q23;5q35) generates the NPM/ALK oncogenic fusion protein associated with ALCL.1 1 Rabbitss, T. H. Nature, 1994, 372, 143
Large cell lymphomas represent about 25% of all non-Hodgkin's lymphomas; about one-third of these tumors are anaplastic large cell lymphoma (ALCL). In turn, more than half the patients with ALCL possess a chromosomal translocation that leads to the in-frame juxtaposition of the 5′ portion of the nucleophosmin (NPM) gene with the sequence encoding for the catalytic domain of ALK kinase. The resulting chimaeric gene, under the control of the strong NPM promoter, drives the expression of the NPM/ALK oncogenic fusion protein. An additional 10% of ALCL patients carry other ALK fusion proteins. To date, 11 ALK fusions have been described. In all cases, the ALK kinase domain sequence is fused to an aminoterminal protein-protein interaction domain of a protein that is highly expressed in the target cell. Thus, the fusion partner provides constitutive expression (through its promoter) and activation (via oligomerisation). In addition, ALK fusion proteins show anomalous cellular localisation. For example, NPM/ALK is mainly found in the cytoplasm and the nucleus. By contrast, wild-type ALK is a tightly regulated, integral membrane protein that is only activated in the presence of a specific extracellular ligand. ALK is normally expressed in the nervous system during embryonic development and is strongly down-regulated at birth, resulting in barely detectable levels in adult tissues. It has been extensively demonstrated that constitutively active NPM/ALK is a potent oncogene with transforming and tumourigenic properties.2 Moreover, rearrangement of ALK kinase is a very early event in tumour formation and is necessary for survival of transformed cells. The high level of expression of NPM/ALK and other ALK fusion protein variants in lymphoma cells and their direct role in lymphomagenesis, combined with the fact that normal ALK is expressed at low levels in the human body, suggests that ALK could potentially be an ideal target for therapy. 2 Morris, S. W; Kirstein, M. N.; Valentine, M. B.; Dittmer, K. G.; Shapiro, D. N.; Saltman, D. L.; Look; A. T. Science, 1994, 263, 1281-1284
Chronic Myeloid Leukemia (CML) is a myeloproliferative disease, characterized by the presence of a modified chromosome, named Ph-chromosome. In the eighties, the molecular defect associated with this cytogenetic abnormality was identified and it was established that the Ph-chromosome results from the chromosomal rearrangement t(9q34;22q11) and leads to the formation of the hybrid gene BCR-ABL coding for the oncogenic Bcr-Abl fusion tyrosine kinase associated with CML and ALL3. In the late 1980s, the data accumulated on the role of BCR-ABL in onset and progression of CML indicated BCR-ABL as the most attractive target for molecularly targeted therapy approaches. Therefore attempts to inhibit the TK activity of the oncoprotein were initiated and this process finally led to the discovery and the development of imatinib mesylate. Imatinib has been under clinical investigation for almost 8 years (50.000 patients) with remarkable results in terms of durable remissions. During the successful clinical trials, resistance to imatinib emerged particularly in patients with acute leukemias, but it is a potential issue also in patients in chronic phase. The molecular mechanism of resistance has been identified in Bcr-Abl gene amplification and mutations in the catalytic kinase domain of the gene3. Mutations render the target kinase insensitive to the drug, either by altering the conformational equilibrium of the catalytic domain, or by changing the drug binding site. This has prompted intense research to find new compounds able to overcome the resistance problem, such as Dasatinib4, Bosutinib5 and Nilotinib.6 These second generation inhibitors show increased potency compared to imatinib and are able to target most of imatinib-resistant clones. However, none of them is able to inhibit efficiently the imatinib-resistant Bcr-Abl T315I mutant. Thus, under the selective pressure of molecularly targeted therapies, the mutation of the gatekeeper amino acid threonine into an isoleucine (T315I) has evolved as the predominant one in patients3 and has proved to be critical for the resistance of the tumour towards Bcr-Abl kinase selective therapies'. These facts indicate that the T315I mutant is a crucial target for the development of new selective therapies aimed at eradicating the disease. 3 Ben-Neriah, Y., Daley, G. Q., Mes-Masson, A. M., Witte, O. N. & Baltimore, D. Science. 1986, 233, 2124 Shah, N. P., Tran, C., Lee, F. Y., Chen, P., Norris, D. & Sawyers, C. L. Science 2004 305, 399-4015 Puttini, M.; Coluccia, A. M.; Boschelli, F.; Cleris, L.; Marchesi, E.; Donella-Deana, A.; Ahmed, S.; Redaelli, S.; Piazza, R.; Magistroni, V.; Andreoni, F.; Scapozza, L.; Formelli, F. & Gambacorti-Passerini, C. Cancer Res. 2006, 66, 11314-113226 Weisberg, E., Manley, P. W., Breitenstein, W., Bruggen, J., Cowan-Jacob, S. W., Ray, A., Huntly, B., Fabbro, D., Fendrich, G., Hall-Meyers, E., Kung, A. L., et al. Cancer Cell. 2005 7, 129-1417 Gambacorti-Passerini, C. B., Gunby, R. H., Piazza, R., Galietta, A., Rostagno, R. & Scapozza, L. Lancet Oncol. 2003, 4, 75-85.
RET (Rearranged during Transfection) proto-oncogene is involved in the onset of hereditary and sporadic thyroid cancer.8 Activating mutations have been described both in the extracellular and the catalytic domain. In addition, rearranged forms of RET have been identified, in which the kinase domain is fused to an activating gene. In all cases, RET kinase activity is switched on independently of ligand binding and induces malignant transformation of thyroid cells. RET uncontrolled activity is both sufficient and necessary to cause neoplastic phenotype. Therefore, it represents an ideal target for molecular therapy of thyroid neoplasias. 8 Jhiang S M. Oncogene 2000, 19:5590-7.
Several small molecule compounds have been described as RET inhibitors during the last few years.9 However, all these compounds were developed against other targets and indeed hit a number of other kinases. This fact is likely to cause significant toxicity in clinical practice. Therefore, RET-selective inhibitors are needed for the management of this group of malignancies. 9 Gunby et al. Anti-Cancer Agents in Medicinal Chemistry 2007, 7, 594.
The disclosed inhibition of ALK, RET, and Bcr-Abl mutant T315I has been demonstrated using an ELISA-based in vitro kinase assay that has been previously developed (EP1454992). Furthermore cellular activity of the compounds on NPM/ALK transformed cells has been demonstrated by tritiated thymidine based cell proliferation inhibition assay.
The inhibitors of the present invention have the following formula or pharmaceutical acceptable salts thereof.